There is considerable debate on the health impacts of soil-transmitted helminth infections. We assessed effects of deworming on physical fitness and strength of children in an area in Yunnan, People's Republic of China, where soil-transmitted helminthiasis is highly endemic.2 max estimate) and the number of 20-m laps completed were recorded. Physical strength was determined with grip strength and standing broad jump tests. Body height and weight, the sum of skinfolds, and hemoglobin levels were recorded as secondary outcomes.The double-blind, randomized, placebo-controlled trial was conducted between October 2011 and May 2012. Children, aged 9–12 years, were treated with either triple-dose albendazole or placebo, and monitored for 6 months post-treatment. The Kato-Katz and Baermann techniques were used for the diagnosis of soil-transmitted helminth infections. Physical fitness was assessed with a 20-m shuttle run test, where the maximum aerobic capacity within 1 min of exhaustive exercise (VOTrichuris trichiura-infected children had 1.6 ml kg−1 min−1 (P = 0.02) less increase in their VO2 max estimate and completed 4.6 (P = 0.04) fewer 20-m laps than at baseline compared to non-infected peers. Similar trends were detected in the VO2 max estimate and grip strength of children infected with hookworm and Ascaris lumbricoides, respectively. In addition, the increase in the VO2 max estimate from baseline was consistently higher in children with low-intensity T. trichiura and hookworm infections than in their peers with high-intensity infections of all soil-transmitted helminths .Children receiving triple-dose albendazole scored slightly higher in the primary and secondary outcomes than placebo recipients, but the difference lacked statistical significance. T. trichiura infections on physical fitness warrants further investigation.We found no strong evidence for significant improvements in physical fitness and anthropometric indicators due to deworming over a 6-month follow-up period. However, the negative effect of  Children from the developing world are often burdened with intestinal worms due to poor water supply, sanitation, and hygiene. However, the assessment of the burden due to intestinal worms is difficult, and thus, the benefits of deworming are unclear. In this study, we determined the effect of deworming on the physical fitness and strength of 9- to 12-year-old children in Yunnan, China, where intestinal worms are common. Children were treated with triple-dose albendazole or placebo and monitored over a 6-month period. Stool samples were collected for the diagnosis of intestinal worm infections. Physical fitness was estimated with a 20-m shuttle run test and physical strength was assessed with grip strength and standing broad jump tests. Children receiving triple-dose albendazole scored slightly higher values in the primary and secondary outcomes than those children who were given placebo. However, the differences were not significant. We also found that children infected with intestinal worms performed significantly worse in the physical fitness and strength tests than their non-infected counterparts. In particular, the negative impact of whipworm infection on physical fitness warrants further investigation. Ascaris lumbricoides, Trichuris trichiura, and the hookworms , are the most common parasitic worm infections of humans. Indeed, more than 1 billion people are infected and approximately 5.4 billion people are at risk of infection Soil-transmitted helminths, namely The global burden of soil-transmitted helminthiasis is currently estimated at 5.2 million disability-adjusted life years (DALYs), mainly due to sub-clinical morbidities, but also anemia and reduced cognitive and physical development T. trichiura and hookworm infections in two cross-sectional studies Based on the rationale that lowering infection intensity would help to control morbidity associated with chronic helminth infection, and that morbidity is infection intensity-dependent, the World Health Organization (WHO) advocates periodic deworming of at-risk populations  with single-dose albendazole (400 mg) or mebendazole (500 mg) We designed a randomized controlled trial to investigate the health benefits of deworming and thereby deepen our understanding of the burden caused by soil-transmitted helminth infection among school-aged children. The study was conducted in a highly endemic area in the People's Republic of China (P.R. China) and assessed the effects of triple-dose albendazole on physical fitness and strength of initially soil-transmitted helminth-infected children. The infection and fitness dynamics were then studied over a 6-month period post-treatment. Changes in anthropometric indicators and hemoglobin levels were also measured, and are reported as secondary outcomes.The study protocol was approved by the institutional research commission of the Swiss Tropical and Public Health Institute . The ethics committee of Basel  and the Academic Board of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention  provided ethical clearance. The trial is registered with Current Controlled Trials (identifier: ISRCTN 25371788).Strongyloides stercoralis were offered a single dose of ivermectin (200 µg/kg).The village doctor, chief, and teachers of each village were briefed on the aims of the study. With help from the teachers, the investigators further explained the procedures to the children and their parents/guardians. Written informed consent was obtained from parents/guardians, whereas children assented orally. Data were kept anonymous. After the 6-month final follow-up, all children attending the five schools were given triple-dose albendazole (3×400 mg) irrespective of their infection status, study participation, and treatment during the study. Children diagnosed with Participants were recruited from five primary schools, where a 70% or higher prevalence of soil-transmitted helminth infections had been detected during a rapid appraisal. All schools belonged to villages exclusively inhabited by the Bulang ethnic minority group, and were located in the mountainous Bulangshan township bordering Myanmar, a sub-division of Menghai county in Xishuangbanna Dai autonomous prefecture, situated in Yunnan province, P.R. China. The five villages are: (i) Sandui ); (ii) Kongkan ; (iii) Laozhai ; (iv) Laonandong ; and (v) Mannuo . Prior to the current trial, no survey or control activities targeting soil-transmitted helminthiasis had been implemented in the study villages. Detailed information on the study area has been published along with data on soil-transmitted helminth re-infection patterns among participants −1 min−1 difference in the maximum aerobic capacity within 1 min of exhaustive exercise (VO2 max estimate) between the intervention and placebo groups.The study was designed as a double-blind, randomized, placebo-controlled trial with three follow-ups, and was carried out between October 2011 and May 2012. Assuming a prevalence of 70% with any soil-transmitted helminth infection and 50% loss to follow-up, the trial aimed to enroll 250 children at baseline to achieve a power of 80% at an alpha error of 5% for the detection of a 2.5 ml kgInclusion criteria for the trial were: (i) provision of two stool samples at baseline; (ii) presence of at least one type of soil-transmitted helminth infection; (iii) no deworming treatment within 6 months before the current study; (iv) no known allergy to albendazole; (v) no major systemic illnesses as determined by a medical doctor; (vi) no concurrent participation in other clinical trials; (vii) residency in the study area for at least 1 year before enrolment; and (viii) participant should be between the age of 9 and 12 years.Children aged 9–12 years who met the inclusion criteria were enrolled by field investigators for a baseline assessment involving parasitological examination, physical fitness and strength tests, and anthropometric and hemoglobin measurements. The same measurements were repeated 1, 4, and 6 months after treatment, with the exception of anthropometric indicators that were only re-assessed at the 4- and 6-month follow-ups .The treatment allocation sequence was generated by a statistician using block randomization with randomly varying block sizes of 2, 4, and 6. Albendazole and placebo tablets were packaged by staff not involved in the field work into sealed envelopes marked with unique identifiers. Following the order of the class list provided by the teachers, each child was sequentially assigned a random number, which corresponded to a number on the sealed envelopes. Both children and field investigators were blinded to the nature of the tablets. The assigned triple-dose treatment  or 3× shape- and color-matched placebo ), was started on treatment day 1 with a single dose, with subsequent doses administered every day until treatment day 3. The field investigators directly observed the consumption of each treatment by all children.A. lumbricoides, hookworm, and T. trichiura, and Baermann for larvae of S. stercoralisTaenia spp. proglottids. Using the Kato-Katz technique, eggs of A. lumbricoides, hookworm, and T. trichiura were counted separately and the results of both slides averaged. The mean was then multiplied by a factor of 24 to obtain the number of eggs per 1 g of stool (EPG). For quality control purposes, the two Kato-Katz slides were examined independently and results compared. Slides were re-read if inconsistencies were detected. An inconsistency was defined as a difference in the infection intensity (EPG) groupings based on WHO guidelines Two stool samples were collected from each child on consecutive days. Both the Kato-Katz (duplicate slides per sample) and Baermann techniques (one examination per sample) were used; Kato-Katz for the detection of eggs of 2 max estimate (to the nearest 0.1 ml kg−1 min−1) with an equation put forth by Léger et al.Physical fitness was assessed with a 20-m shuttle run test Physical strength was assessed with a grip strength and a standing broad jump test. For the grip strength test, the hand span (distance from the tip of the thumb to the tip of the little finger) of the child's dominant hand was measured (to the nearest 0.5 cm) and an electronic dynamometer  adjusted accordingly to provide the optimal grip span 2; the BMI-for-age Z score (BAZ) and height-for-age Z score (HAZ) were used as indicators for wasting and stunting, respectively −1, with a HemoCue Hb 301 system  using a drop of blood from the ear lobe. Anemia was defined according to WHO age- and sex-specific cut-offs For the measurement of body height and weight, children were asked to take off their shoes and sweater before standing on a digital weighing scale  or stadiometer  The socioeconomic status of the participants at baseline was assessed through a questionnaire asking the children for the education level of their parents and the main source of their household income. The full trial protocol is available as supporting information .Data were entered into Excel version 2008 , double-checked, and merged into a single database for statistical analysis with STATA version 10.0 . The randomization code was broken after data entry and a series of internal consistency checks were completed. A per-protocol analysis was carried out in an un-blinded manner.In the primary analysis, physical fitness and strength scores, anthropometric measurements, and hemoglobin levels were expressed as means, and changes in the means between baseline and treatment follow-ups were compared between treatment groups in a multivariate linear regression model. In a sub-analysis, changes in the means of physical fitness and strength indicators between baseline and follow-up were compared among children of distinct soil-transmitted helminth infection status regardless of treatment status. To further explore the effect of infection intensity on these measurements, distinct groups of children were identified using principal component and cluster analysis, based on species-specific soil-transmitted helminth log transformed egg counts at baseline and at the 1- and 4-month follow-ups. Changes in the means of physical fitness and strength indicators between baseline and follow-up were compared among six biologically meaningful groups of varying soil-transmitted helminth infection intensity.As illustrated in 2 max estimate and number of 20-m laps completed were 44.9 ml kg−1 min−1 (standard deviation (SD): 2.8 ml kg−1 min−1) and 23.8 laps (SD: 8.7 laps), respectively, for the albendazole group, and 45.4 ml kg−1 min−1 (SD: 3.2 ml kg−1 min−1) and 25.2 laps (SD: 9.9 laps), respectively, for the placebo group. With regard to physical strength, the mean grip strength and standing broad jump distance were 12.6 kg (SD: 4.1 kg) and 142 cm (SD: 14 cm), respectively, for the albendazole group, and 12.6 kg (SD: 3.5 kg) and 142 cm (SD: 14 cm), respectively, for the placebo group. However, when further stratified by sex, boys had higher physical fitness and strength than girls   .−1 (SD: 24 g l−1) and 157 g l−1 (SD: 28 g l−1), respectively.Treatment groups were also comparable in terms of anthropometric and hematologic characteristics at baseline. Despite the mean BMI for the albendazole and placebo group being 16.0 (SD: 1.3) and 16.1 (SD: 1.3) respectively, the baseline prevalence of wasting was only 3.6%. On the other hand, stunting was present in 76.8%  and 126.3 cm (SD: 7.1 cm), respectively) of the cohort. Baseline prevalence of anemia was low at 4.6%, as the mean hemoglobin levels for the albendazole and placebo group were 161 g l2 max estimates increased by 1.0–2.3 ml kg−1 min−1 from baseline over the 6-month trial period for the albendazole group, while the increase for the placebo group ranged from 0.2–2.1 ml kg−1 min−1. Likewise for the number of 20-m laps completed, the range of increase was 3.4–11.9 laps and 1.4–11.7 laps for the albendazole and placebo group, respectively. When adjusted for village, and at the individual level for sex, age, height, and weight at follow-up, the difference in the increase of physical fitness between both groups was highest at the 1-month follow-up, where the increase from baseline in the albendazole group was 0.9 ml kg−1 min−1 (P = 0.05) or 2.1 laps (P = 0.14) higher than the placebo group. With regard to physical strength, the grip strength increased 0.8–2.0 kg from baseline for the albendazole group, while the placebo group experienced an increase of 0.4–1.8 kg. The difference in the increase between both groups was highest at the 1-month follow-up , but this difference was not statistically significant. The largest change in standing broad jump distance from baseline was observed among the albendazole group at the 1-month follow-up (+2 cm), but the placebo group fared better at the 6-month follow-up (+2 cm). However, both of these increases were not statistically significant.Children receiving triple-dose albendazole experienced a greater, but mostly not statistically significant, change in the means of their physical fitness scores than their peers from the placebo group at all three follow-ups . VO2 max−1 (P = 0.72) and 3 g l−1 (P = 0.49) higher compared to the placebo group On the other hand, the increase from baseline in the albendazole group was 3 g l−1 higher than the placebo group at the 4-month follow-up (P = 0.65).In terms of secondary outcomes, children in the albendazole group had a larger increase, from baseline, in the means of their body height and weight and sum of skinfolds than their counterparts from the placebo group . The ranT. trichiura-infected children had 1.6 ml kg−1 min−1 less increase in their VO2 max estimate from baseline than their non-infected peers at the 1-month follow-up (P = 0.012). Similarly, hookworm-infected children had 1.1 ml kg−1 min−1 less increase in their VO2 max estimate from baseline than their non-infected peers at the 6-month follow-up (P = 0.03). In addition, the increase in the number of 20-m laps completed from baseline was 4.6 (P = 0.04) and 6.0 (P = 0.01) laps less for T. trichiura-infected children than their non-infected counterparts at the 1- and 4-month follow-ups, respectively. As further illustrated in T. trichiura infection intensity than diminished A. lumbricoides and hookworm infection intensity. In terms of grip strength at the 1-month follow-up (A. lumbricoides-infected children was 0.8 kg lower than among children not infected with this helminth species (P = 0.05), but hookworm-infected children had 0.9 kg more increase from baseline than their non-infected peers (P = 0.04). No statistically significant change in standing broad jump distance due to soil-transmitted helminth infection status was observed at each of the three follow-ups.When the soil-transmitted helminth infection status was used as explanatory variable for the primary outcomes , T. tricollow-up , the incA. lumbricoides re-infection by the 4-month follow-up, high infection intensity of T. trichiura at all time-points, and no or minimal hookworm re-infection at follow-ups; group 4, low intensity of A. lumbricoides re-infection by the 4-month follow-up, intermediate infection intensity of T. trichiura at all time-points, and no or minimal hookworm re-infection at follow-ups; group 5, intermediate intensity of A. lumbricoides re-infection by the 4-month follow-up, and no or minimal T. trichiura and hookworm re-infection at follow-ups; and group 6, infection intensity of all species were higher during the follow-ups compared to the pre-treatment baseline. When group 1 was used as the reference group in the multivariate linear regression models . A similar trend was observed for the number of 20-m laps completed and a statistically significant 5.7 more 20-m laps were completed by children from group 5 as compared to group 1 (P = 0.04) and the baseline. In terms of standing broad jump distance, children from group 4 had a 6 cm higher increase from baseline than children from group 1 at the 4-month follow-up (P = 0.03). No statistically significant change in grip strength dependent on soil-transmitted helminth infection intensity was observed at all follow-ups.When the children were grouped according to their longitudinal infection intensity patterns, the six groups that emerged  had the n models , childreT. trichiura, A. lumbricoides, and hookworm at 94.5%, 93.3%, and 61.3%, respectively. Therefore, an intensive de-worming regimen, consisting of triple-dose albendazole A. lumbricoides occurred more rapidly than expected and the prevalence of A. lumbricoides reached 80% of the pre-treatment prevalence 4 months after treatment T. trichiura, corroborating previous conclusions that T. trichiura infection is particularly hard to cure with current anthelmintic drugs As shown in our preceding work in Bulang communities 2 max estimate at 1-month follow-up between the albendazole and placebo groups despite the relatively small sample size. It is interesting to note the variation in hemoglobin levels for both groups throughout the follow-up period and this could probably be due to seasonal dietary changes or the presence of other infections. In addition, we observed a general learning effect with the physical fitness and strength tests, especially the 20-m shuttle run test, amongst the children, but this was mitigated in the analysis by having a control group.In a recently published trial conducted in India T. trichiura-infected children performed worse in the 20-m shuttle run than their non-infected peers. This confirmed the results from a cross-sectional study conducted by our group where T. trichiura-infected children were found to complete, on average, 6.1 20-m laps less and have a VO2 max estimate which was 1.9 ml kg−1 min−1 lower than their non-infected counterparts T. trichiura worms anchor their whip-like anterior end into the wall of the large intestine and caecum by secreting pore-forming proteins. Such an invasive mechanism causes inflammation and bleeding, resulting in abdominal pain in the short term, and anemia and rectal prolapse in the long term, especially when large numbers of worms are present T. trichiura might enhance the host's endurance in exhaustive exercises, such as the 20-m shuttle run. Hookworm-infected children were also found to have a significantly lower increase from baseline in their VO2 max estimates than children not infected with hookworm at the 6-month follow-up. Although anemia is a known symptom of hookworm infection and would be a plausible cause for reduced VO2 max estimates A. lumbricoides undergo a similar migratory process 2 max estimates was observed in children infected with A. lumbricoides. In terms of grip strength, the increase from baseline among A. lumbricoides-infected children was significantly lower, while hookworm-infected children had a higher increase from baseline, when compared to their non-infected peers. As there is currently limited evidence on the association of soil-transmitted helminth infection and grip strength, these inconsistent findings warrant further investigation.In the sub-group analysis, we found that soil-transmitted helminth-infected children had performed significantly worse in the physical fitness and strength tests than their non-infected peers. When we grouped children according to their infection status at each follow-up, we observed that T. trichiura and hookworm re-infection achieved higher improvements during the follow-ups in the 20-m shuttle run, as compared to peers with high infection intensity of all species. In addition, children with no or minimal A. lumbricoides and hookworm re-infection performed better in the standing broad jump than their counterparts with high infection intensity of all species. These findings provide further evidence of the impact of soil-transmitted helminth infections on the physical fitness and strength of school-aged children.When children were grouped according to infection intensity, we were able to take into consideration the degree of infection at baseline, 1-month, and 4-month follow-ups, and the extent of multiparasitism for each child. These analyses revealed that individuals with a combination of no or minimal The anthropometric and physical strength findings from this trial should be viewed in the light of the following limitations. A follow-up period of 6 months is probably too short for an accurate evaluation of anthropometric gains and physical strength increments from longer-term physical growth due to deworming. Taking into account that keeping controls untreated for a long period would be difficult due to ethical considerations, a 3- to 5-year prospective cohort study, where children are treated regularly to ensure that they are helminth-free, and the changes in anthropometric indicators and physical strength from baseline are monitored and compared with changes in soil-transmitted helminth infection intensity over time, could be a more appropriate study design. Finally, catch-up growth after anthelmintic treatment can only occur if the diet is sufficient A. lumbricoides and low cure rates with T. trichiura. However, negative impacts on the physical fitness and strength were observed in school-aged children infected with soil-transmitted helminths in sub-group analyses. In particular, the clear effects of T. trichiura infection on physical fitness in this trial is intriguing as the public health burden of this helminth species is currently not as well defined as that of the other two species. The fact that T. trichiura infection had the strongest negative impact on the physical fitness of the children but was hardly cured with triple-dose albendazole is another major concern. Finally, we also showed that the observed morbidities were infection intensity-dependent and in order to control them, regular deworming, coupled with dietary improvements and water, sanitation, and hygiene development, should be considered.We conclude that there is no strong evidence for significant improvements in physical fitness and anthropometric indicators due to deworming with triple-dose albendazole. This might be partly explained by the rapid re-infection observed with Checklist S1CONSORT checklist(DOC)Click here for additional data file.Protocol S1Study protocol for trial entitled “Effect of deworming on physical fitness of school-aged children in Yunnan, China: a double-blind, randomized, placebo-controlled trial.”(DOC)Click here for additional data file.


Salt-sensitive individuals show earlier and more serious cardiac damage than nonsalt-sensitive ones. Some studies have suggested that microRNA-133a could reduce cardiac hypertrophy and myocardial fibrosis. The current study aims to investigate the different functions of high-salt intake on salt-sensitive (SS) rats and Sprague-Dawley (SD) rats and the involvement of microRNA-133a in these roles. After high-salt intervention, the left ventricular mass (LVW) and left ventricular mass index (LVMI) of the salt-sensitive high salt (SHS) group were obviously higher than those of the salt-sensitive low salt (SLS) group. However, the difference between the Sprague-Dawley high salt (DHS) group and the Sprague-Dawley low salt (DLS) group was not significant. Compared with SLS group, collagen I and connective tissue growth factor (CTGF) in the heart of SHS group were significantly higher, whereas no statistical difference was observed between the DHS group and the DLS group. Compared with low-salt diet, microRNA-133a in the heart of both strains were significantly decreased, but that in the SHS group decreased more significantly. These results suggest that high salt intervention could down-regulate the expression of myocardial microRNA-133a, which may be one of the mechanisms involved in myocardial fibrosis in salt-sensitive hypertension. Hypertension is a primary risk factor for cardiovascular events, and salt-sensitivity is an intermediate genetic phenotype of essential hypertension. High salt intake can elevate blood pressure, and directly result in heart , brain  and kidnet al. [Over the years, much research attention has been directed toward microRNAs. MicorRNAs comprise a 22-nucleotide-long RNA sequence that can regulate protein synthesis by binding to the untranslated regions of targeted mRNAs, thereby altering stability of the target protein . Studieset al.  demonstret al. , connectet al.  and collet al.  in diffeThe current study aims to observe the influence of high-salt intake on blood pressure, left ventricular weight, myocardial microRNA-133a and fibrosis levels by implementing high-salt diet intervention trial in Dahl salt-sensitive rats and SD rats. The final objective is to discuss the role of microRNA-133a in salt-sensitive hypertensive cardiac fibrosis with high-salt intervention.p > 0.05). After high-salt intake, SBP increased significantly in salt-sensitive (SS) rats and in Sprague-Dawley (SD) rats . The increment of SBP in SS rats after high-salt intake was more significant (p < 0.01) compared with that in SD rats  in the same strain of rats ( SD rats .p < 0.01), whereas no significant difference was observed in each group of SD rats.As shown in p < 0.01) (p > 0.05), but collagen I mRNA expression in SHS group was significantly higher than that in SLS group . Collagen I mRNA expression was similar in DHS group and in DLS group  (Masson’s stain was used to show the collagen fiber that was stained blue in the myocardium. As shown in  < 0.01) A. By con > 0.05) B.p < 0.01) . No statistical difference was observed in the DHS group and the DLS group   A. CTGF m > 0.05) B.vs. 0.99 ± 0.25, p < 0.01; SD: 0.53 ± 0.14 vs. 1.00 ± 0.20; p < 0.01). Compared with the DHS group, the decline of microRNA-133a in the SHS group was more significant (p < 0.05)  .High-salt intake is an important environmental factor that may lead to essential hypertension. It can result in higher cardiovascular risk, myocardial hypertrophy and fibrosis, as well as vascular remodeling independently of the blood pressure . InvestiIn the current study, results indicate that compared with low-salt diet, high-salt leads to blood pressure increment in the two strains and the BP in SS rats increased more significantly. After high-salt intervention, LVM and LVMI of the SS rats were obviously higher than those of the SD rats. Other studies have confirmed that salt-sensitive hypertension entails more serious cardiac hypertrophy and fibrosis and that high salt could directly lead to cardiac fibrosis independently of blood pressure . It suggStudies have shown that microRNAs are involved in many kinds of pathophysiologic process and both microRNA-133a and microRNA-1 are involved in cardiac hypertrophy and fibrosis . ExpressAfter high-salt intervention, microRNA-133a in the SD rats was suppressed by nearly 50%, but the expression of cardiac CTGF and collagen I did not increase correspondingly. For our analysis, compared with Dahl SS rats, SD rats were insensitive to high-salt intervention, a period of 4 weeks of high-salt intervention could suppress the expression of microRNA-133a, but it is not enough to cause an increase in myocardial collagen I in SD rats. Perhaps extending the high-salt intervention time will lead to an increase in the expression of CTGF and collagen I.AngII was greatly responsible for myocardial fibrosis [AngII concentration in myocardium, and this effect was independently of its effect on blood pressure [AngII could suppress microRNA-133a expression via AT1 receptor. In the present study, we found that the expression of microRNA-133a was down-regulated both in Dahl SS rats and in SD rats, so we speculate that high-salt was likely to down-regulated microRNA expression via its effect on RAAS system in heart tissue. On the other hand, SBP of the SD rats was slightly elevated after high-salt intervention, so it indicated that high-salt intake could disturbed the expression of microRNA133a independently of its effect on blood pressure. But it is just a hypothesis and there need to be more experiments to verify it. Deleting the of AT1 receptor gene of Dahl SS rats or supplementing AT1 receptor antagonist to them may be viable methods.Activation of the local Renin–Angiolensin–Aodosterone System (RAAS), especially the concentration of fibrosis ,22. Highpressure ,24. Recepressure  showed tTwo kinds of rats were included in this study, namely, Dahl salt-sensitive (SS) rats  and Sprague-Dawley (SD) rats . Sixteen SS rats aged 4 weeks were randomly divided into two groups. The SS high-salt (SHS) group received 8% NaCl diet and the SS low-salt (SLS) group received 0.4% NaCl diet. Sixteen SD rats aged 4 weeks received the same treatment as the SS rats. The two groups were designated as SD high-salt (DHS) group and SD low-salt (DLS) group respectively. The intervention lasted for 4 weeks. The rats were reared in the specific pathogen free (SPF) animal room and had free access to food and water. The illumination time was 12 h/day. The institutional ethics committee of Xi’an Jiaotong University Medical School approved all experiment protocols .Blood pressure was measured at the beginning and at the end of intervention. BP2000 animal noninvasive blood pressure measuring instrument  was used to determine tail systolic blood pressure. Rats received anesthesia via intra-peritoneal injection of 10% chloral hydrate. Rat hearts were acquired and the left and right atriums were cut along the atrioventricular ring. The free wall of the right ventricular was also cut along the interventricular septum. The left ventricular mass (LVW) was weighed and the left ventricular mass index (LVWI = LVW/body weight (mg/g)) was calculated. About 1/3 of the left ventricular myocardium was immersed in 4% formalin for 48 h and then embedded with paraffin, whereas 2/3 was preserved in an −80 °C refrigerator and used for RNA extraction.The formalin-fixed paraffin-embedded heart tissue was stained with Masson’s trichrome (MT). For evaluation of cardiac fibrosis, the MT staining pictures were taken randomly under light microscopy (×400) from 10 different microscopic fields per section by digital camera . Data are reported as the mean collagen volume fraction (CVF) in each field area. The CVF was determined by quantitative morphometry using an Image-Pro plus software .2O2 in methanol, followed by a rinse with phosphate buffered saline (PBS). All sections were treated with 10% blocking solution for 30 min to prevent nonspecific antibody binding. They were then incubated overnight at 4 °C with primary antibody. After rinsing in PBS, sections were then incubated for 30 min at 37 °C with secondary antibody followed by a rinse with PBS. Then sections were treated with SP at 37 °C for 30 min. Slides were stained with DAB, counterstained in hematoxylin, dehydrated, and mounted. Control immunostaining was carried out by the same procedure in which the first antibody was replaced by PBS. Sections were photographed with a fluorescence microscopic imaging system . To analyze the expression of CTGF, ten non-overlapping high-power fields (magnification 400×) were randomly selected from each cardiac section. Data are reported as the mean optical density in each field area.The myocardial CTGF expression was detected through immunohistochemical methods. The antibody was from Biosynthesis Biotechnology Co., Ltd. . Immunostaining for CTGF in cardiac tissue was examined by strept avidin–biotin complex (SABC) method. Briefly, sections were deparaffinized with xylene and then dehydrated with ethanol. Endogenous peroxidase activity was inhibited for 10 min with 0.3% HCTGF and collagenI mRNA detection and U6 was used as internal standard for microRNA-133a detection. The primers used were 5'-TCGTGGAAGGGCTCATGACC-3' (sense) and 3'-TGACCTTGCCCACAGCCTTG-5' (antisense) for GAPDH, 5'-TGCCTACCGACTGGAAGACA-3' (sense) and 3'-TGGCAGGCACAGGTCTTGAT-5' (antisense) for CTGF, 5'-GGCGAAGGCAACAGTCGATT-3' (sense) and 3'-TTCCGAATTCCTGGTCTGGG-5' (antisense) for collagen I, 5'-ATTGGAACGATACAGAGAAGA-3' (sense) and 3'-GGAACGCTTCACGAATTTG-5' (antisense) for U6. The results were normalized to the DLS group.The myocardial tissue microRNA-133a, CTGF, and collagen I were detected through real-time PCR. The kits were from Takara Bio Inc. . GAPDH was used as internal standard for t test was utilized to determine the difference in parameters between the rats that received different diets. Two-tailed p < 0.05 was considered statistically significant.Data are presented as mean ± SD. Comparison of the blood pressure before and after the intervention was conducted through analysis of variance with the repeated measure design. Student’s This study demonstrates that high-salt intake could suppress the expression of myocardial microRNA-133a partly independent of its effect on blood pressure. Myocardial down-regulation microRNA-133a may represent a regulatory mechanism that leads to myocardial fibrosis. The findings of the present study suggest that up-regulation of microRNA-133a may suppress myocardial fibrosis in salt-sensitive hypertension.


In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40–80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for  However, the use of viral vectors carries significant biological risks such as uncontrolled activation of immunogenic response, carcinogenesis, broad tropism and difficulty of vector production  is suitable for detecting and quantifying free hydroxyl radicals generated by the collapse of cavitation bubbles obtained with ultrasound irradiation. During this process, the TA solution (20 mM of TA in PBS) as a dosimetric solution reacts with a hydroxyl radical generated through water sonolysis. Therefore, 2-hydroxyterephthalic acid (HTA) is produced which can be detected using spectroscopy with excitation and emission wavelengths of 318 and 426 nm, respectively .To examine the effect of ultrasound on plasmid integrity, 2 ml tubes containing 10 μg of pEGFP-C1 in 650 μl of Opti-MEM  were separately exposed for 5, 10, 20, 30, 60 and 80 seconds at a CI of 12. In parallel, we also tested different CI values  for 30 seconds. After ultrasound exposure the integrity of each plasmid sample was examined by agarose-ethidium bromide electrophoresis. The result was compared with control pEGFP-C1 which had not undergone ultrasonic exposure.6 cells/ml, in order to achieve 0.5–0.8.106 cells/ml on the day of sonoporation. Adherent cells were plated at 2.106 cells per 75 cm2 in order to achieve approximately 70% confluence after 48 hours. Prior to ultrasound exposition, non-adherent or trypsinized adherent cell lines were harvested and centrifuged  then washed twice with Opti-MEM to remove serum traces, a step which significantly improves transfection efficiency [6 or 10.106 cells/ml in Opti-MEM were placed in 2 ml tube and pEGFP-C1 plasmid was added to cell suspensions at the indicated concentration. After ultrasound application, cell suspensions were placed in complete medium in a 6-well cell culture plate and incubated at 37°C in humidified atmosphere with 5% CO2 incubator for 24 hours.Forty-eight hours prior to transfection, non-adherent cells were seeded at a concentration of 0.2–0.4.10ficiency . 550 μl 6 cells/ml in 100 μl Nucleofactor Solution V (Cell Line Solution V Nucleofector Lonza) at room-temperature. Two micrograms of pEGFP-C1 vector were added to the cell suspension. The cell / DNA mixtures were nucleoporated in 1 cm transfection cuvettes according to a predefined program on the Nucleofactor. Twenty-four hours post-nucleofection the transfection efficiency and cell viability were evaluated by flow cytometry after propidium iodide (PI) staining . Lipofection using Lipofectamine 2000 Transfection Reagent  was conducted on the Jurkat and K562 cell lines according to the manufacturer’s instructions.We performed a study to compare the transfection efficiency and the cell viability of sonoporation with those of two commercially available transfection methods: nucleofection and lipofection. This study was performed on two non-adherent cell lines, Jurkat and K562. For nucleofection transfection, the procedure was performed according to the manufacturer’s recommendations . Briefly, Jurkat and K562 cells were pelleted by centrifugation at 90g and 200g for 10 minutes, respectively, and resuspended at a density of 10.10In this study the plasmid peGFP-C1, encoding GFP under the control of a CMV promoter was employed to monitor transfection. Twenty-four hours after sonoporation of peGFP-C1 vector, GFP-positive cells were observed using an Olympus IX50 microscope at a excitation wavelength of 488 nm. The transfection efficiency and cell viability were monitored using a flow cytometer (fluorescence-activated cell sorter) (FACS) . Adherent cells were harvested by trypsinisation as well as non-adherent cells present in the culture medium. The cell suspension was centrifuged  then washed twice in PBS. In order to assess cell viability, PI was added to cells for 10 min according to the manufacturer's recommendations prior to FACS analysis. During FACS analysis the gate was drawn to exclude debris. Results were expressed as a percentage of GFP positive cells using BD Biosciences FACS Diva software. This percentage was calculated on the basis of the viable cells.To determine reproducibility, three independent experiments were conducted (each performed at least in triplicate). All results are reported and displayed as mean ± standard deviation. Significance tests were performed using a non-parametric Wilcoxon test . Significance was defined as p < 0.05.The inertial cavitation was characterized by a chemical dosimeter TA for OH radicals. The resulting hydroxyterephthalate fluorescence is adequately correlated with the cavitation index  which coet al.[et al. [Maintaining plasmid integrity is an important issue when considering transfection by sonoporation. To examine whether ultrasound affects plasmid integrity, pEGFP-C1 was exposed to ultrasound either at a constant CI of 12 for a duration ranging from 0 to 80 seconds or at different CI values  for a constant time of exposure of 30 seconds. Contrary to the results published by Y. Song et al. and in a.[et al. , we founet al. [6, 2.106, 3.106 and 10.106 cells/ml. Flow cytometry analysis of GFP positive cells and cell viability . + T-cells [Fresh AML cells at a concentration of 10.10 T-cells . Moreove T-cells .6 cells/ml using the optimal exposure conditions as described previously (CI of 12 for 30 seconds and 10 μg of pEGFP-C1). Cell viability was determined using PI staining. Results in To test the biological reproducibility of the device, sonoporation of the adherent cell line (BT474) and two non-adherent cell lines (RL and Jurkat) was conducted. Sonoporation was performed at a cell concentration of 10.10Experiments with Jurkat and K562 cell lines were undertaken to compare the transfection efficiency of sonoporation to those of nucleofection and lipofection. Sonoporation and nucleofection were essentially equivalent in terms of DNA delivery and toxicity as determined by flow cytometry analysis and PI staining. Using sonoporation, the Jurkat line showed 77% viability and 42% transfection efficiency, while transfection of K562 gave 84% viability and 49% transfection efficiency. Nucleofection gave results essentially equivalent in terms of toxicity and efficiency . HoweverRL and K562 cell lines were transfected with pEGFP-C1 plasmid and HL-60 cell line was transfected with vectors coding for shRNA against the cN-II nucleotidase (pScN-II) and a control scrambled sequence (pScont). Transfected cells were selected by continuous exposure to 1.2 mg/ml (RL-pEGFP-C1 cells), 1.5 mg/ml (K562-pEGFP-C1cells) and 1 mg/ml (HL60-pScN-II and HL60-pScont cells) of G418. Five to six days after the addition of selection medium the percentage of cells expressing GFP increased, representing the entire culture after 5 weeks of culture. The polyclonal cell cultures were processed using limiting dilution to isolate clones. As shown in in vitro pDNA delivery in adherent and non-adherent cell lines using ultrasound. First, we developed a new ultrasound device wherein acoustic cavitation is stably controlled over time  with a slight impact on technical reproducibility with densities lower than 10.106 cells/ml. Thus, a CI value of 12, a 30 seconds exposure time, 10.106 cells per ml and 10 μg of pEGFP-C1 are considered as the optimal transfection parameters that showed the highest transfection efficiency with minimal cell damage. The results obtained showed high transfection efficiency up to 80% with 75% of viability in the A549 lung epithelial cell line. In comparison, a sonoporation efficiency of 10–38% was previously reported on the same cell type with the combination of ultrasound and micro-bubbles [Over the past few years, several researchers have used ultrasound-induced cavitation as a tool for drug and gene delivery. Transfer of nucleic acids or drugs is attributed to transient holes in the cell membrane produced by ultrasound-induced cavitation , 20. Thever time . As mainr time (. On the -bubbles , 29. Unl-bubbles , 37–38, A major advantage of our apparatus is that high transfection efficiency was obtained without using contrast agents or transfection reagents. Many studies –29 have in vitro gene delivery. Electroporation-based nucleofection technique by Lonza allows high transfection efficiency in hard-to-transfect cells [Lipofection and nucleofection are the main methods used for routine transfection of adherent and non-adherent cell lines. Lipid-mediated gene delivery methods also referred to as lipofection or liposome-based-gene transfection are easy to use but not applicable to all cell types particularly for non-adherent cell line transfection –40. Nuclct cells –43. Howect cells . Our expin vitro delivery for both adherent and non-adherent cell lines. Our results confirm ultrasound as an alternative non-viral technology for the efficient transient transfection of a wide range of different cells including non-adherent cells or fresh human cells, and the preparation of stably transfected cells.An effective control of spatial and temporal acoustic cavitation was achieved in this study. It provides a well-adapted method for low cost routine pDNA


Activation of the receptor for advanced glycation end products (RAGE) axis may have an important role in apoptosis. Glucagon-like peptide-1 (GLP-1) is a gut hormone that has been proposed as a therapeutic target for the treatment of diabetes, and GLP-1 receptor agonists have been reported to protect against myocardial injury associated with diabetes. The aim of the present study was to investigate the cardioprotective mechanism of exendin-4 (EX-4), a GLP-1 receptor agonist, against myocardial cell apoptosis induced by hyperglycemia. Neonatal rat ventricular myocytes were prepared by enzymatic dissociation and then cultured with high levels of glucose (HG) in the presence or absence of EX-4. Cell apoptosis was detected using an annexin V-fluorescein isothiocyanate/propidium iodide kit, and cell viability was measured using an MTT assay. RAGE expression levels and the activity of caspase-3 were assessed by western blot analysis. The results demonstrated that the incubation of myocytes with HG led to a time-dependent activation of RAGE, and the protein expression of RAGE was increased at 6 h and peaked at 24 h (P<0.05). Hyperglycemia was also found to significantly decrease cell viability and increase apoptosis (P<0.05). In addition, EX-4 significantly inhibited hyperglycemia-induced RAGE expression and the apoptosis of myocytes, and improved cell viability in a dose-dependent manner (P<0.05). When the concentration of EX-4 was 10 nM, the myocardial cell viability was significantly improved, and the levels of RAGE expression and apoptosis were significantly decreased compared with those in the HG group in the absence of EX-4 (P<0.05). Therefore, the results from the present study suggest that the cardioprotective effect induced by EX-4, a GLP-1 receptor agonist, against diabetic cardiomyopathy may be associated with the inhibition of RAGE expression. Diabetes causes various cardiovascular complications, for example diabetic cardiomyopathy, which has become the major cause of morbidity and mortality among patients with diabetes. Previous studies have suggested that cardiomyocyte apoptosis has a key role in diabetic cardiac damage in animals and humans –3. Hyperc release, have been demonstrated to be decreased in the hearts of diabetic RAGE-null mice compared with those in wild-type diabetic littermates during myocardial ischemia and reperfusion (The multi-ligand receptor for advanced glycation end products (RAGE), which was first identified as receptor for the advanced glycation end products (AGEs), is a signal transduction receptor belonging to the immunoglobulin superfamily . RAGE iserfusion . TherefoGlucagon-like peptide-1 (GLP-1), a gut hormone secreted in a nutrient-dependent manner, stimulates insulin secretion and inhibits glucagon secretion and gastric emptying . Therefo5/ml in Dulbecco’s modified Eagle medium supplemented with 15% (v/v) fetal calf serum at 37°C and 5% (v/v) CO2. Following incubation in serum for 24 h, the cells were washed and cultured in serum-free medium for 24 h, and the cultures were then subjected to different treatments.Neonatal rat ventricular myocytes were prepared from the hearts of Sprague-Dawley rats (aged between 1 and 3 days) by enzymatic dissociation, as previously described . BrieflyTo determine the effect of glucose on the expression of RAGE, cells were exposed to high levels of glucose  for different time periods . A total of 25 mmol/l D-glucose was used for the HG treatments, compared with 5 mmol/l D-glucose used as the normal control (NG), as previously described  was added  to each well. The cells were incubated for 4 h, and then, following the addition of 100 μl 10% sodium dodecyl sulfate (SDS) and 0.01 N HCl to dissolve the crystals, the cells were incubated for a further 16 h. The absorbance was determined using an automatic microplate reader at a wavelength of 570 nm. The relative cell viability was expressed as a percentage of the cell viability of the control group.The cultured cells were seeded at a density of 1×105 cells/ml) followed by staining with 5 μl annexin V-FITC and 5 μl PI for 15 min at room temperature in the dark. The percentage of cell apoptosis was then determined using flow cytometry with a BD FACSCalibur platform .Apoptosis was detected using an annexin V-FITC/PI kit  in accordance with the manufacturer’s instructions, as previously described . BrieflyCardiomyocytes were lysed using a Protein Complete Lysis kit . The protein concentration in each sample was determined using a Protein Assay kit (Beyotime Institute of Biotechnology) using bovine serum albumin as a standard. For the immunoblot analysis, proteins were separated using SDS-PAGE, and then transferred onto a polyvinylidene fluoride (PVDF) membrane as previously described ,20. The Statistical analysis was performed using SPSS software, version 13.0 . All values are expressed as the mean ± standard deviation. The Student’s t-test was used for between-group comparisons. Welch’s analysis of variance was used for comparisons among groups, and the Student-Neuman-Keuls or Dunnett’s T3 test was used for post-hoc multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.Incubation of myocytes with HG led to a time-dependent activation of RAGE expression compared with that observed in a low-glucose environment, and the protein expression of RAGE was increased at 6 h and peaked at 24 h P<0.05; . HoweverAs shown in According to the results from the MTT assay, the effects of different concentrations of EX-4 (0.1–10 nM) on myocardial cell apoptosis were determined. Flow cytometric analysis demonstrated that EX-4 inhibited the apoptosis of neonatal myocytes induced by HG in a dose-dependent manner P<0.05; .Cleaved caspase-3 is a key executor in the apoptotic process, which is induced by HG. Therefore, in the present study, the expression of caspase-3 was detected using western blot analysis. As shown in As shown in In the present study, it was demonstrated that hyperglycemia increases the expression of RAGE and induces apoptosis in myocytes. In addition, EX-4, a GLP-1 receptor agonist, was shown to protect against myocardial apoptosis in a dose-dependent manner. The inhibitory effect of EX-4 on apoptosis may be via a reduction in caspase-3 activity and the inhibition of RAGE expression. These results indicate that the cardioprotective effect induced by EX-4 during diabetic cardiomyopathy may be associated with the inhibition of RAGE expression.et al . Since this receptor is abundantly expressed in β cells and throughout the gut, heart, vascular smooth muscle cells, endothelial cells, kidney, lung and peripheral nervous system , GLP-1 aet al  demonstrl  during diabetic cardiomyopathy may be associated with the inhibition of RAGE expression. However, there were certain limitations in the present study, as the effects of EX-4 on RAGE expression and apoptosis were only observed following their induction by hyperglycemia. Therefore, the precise mechanisms underlying the observations require further investigation.


Flueggea leucopyrus (Willd.) for treating various cancers including breast cancer. In the present study, the cytotoxicity of this decoction and its effects on Heat Shock Protein (HSP) expression and apoptosis were compared in three breast cancer phenotypes, to scientifically evaluate if a decoction prepared from F. leucopyrus (Willd.) is useful for the treatment of breast cancer.During the past few years, there has been an increasing interest among the Traditional and Folk medical practitioners of Sri Lanka in the use of a decoction prepared from F. leucopyrus decoction was determined by evaluating its effects in MCF-7, MDA-MB-231 and SKBR-3 breast cancer cell lines, and MCF-10A (non-cancerous) breast cell line, by use of the Sulphorhodamine (SRB) assay. The effect of the decoction on HSP gene expression in the above cells was evaluated by (a) Real time reverse transcription PCR (RT-PCR) and (b) Immunofluorescence analysis of HSP protein expression. Effects of the decoction on apoptosis were evaluated by (a) fluorescent microscopic examination of apoptosis related morphological changes and (b) DNA fragmentation (c) Caspase 3/7 assay.Cytotoxic potential of the F. leucopyrus decoction can mediate significant cytotoxic effects in all three breast cancer cells phenotypes  with little effect in the non-cancerous breast cell line MCF-10A (IC50: 570.4 μg/mL). Significant (*P <0.05) inhibitions of HSP 90 and HSP 70 expression were mediated by the decoction in MCF-7 and MDA-MB-231, with little effect in the SKBR-3 cells. Clear apoptotic morphological changes on Acridine orange/Ethidium bromide staining and DNA fragmentation were observed in all three breast cancer cell lines. Caspase 3/7 were significantly (*P <0.05) activated only in MDA-MB-231 and SKBR-3 cells indicating caspase dependent apoptosis in these cells and caspase independent apoptosis in MCF-7 cells.F. leucopyrus mediates cytotoxic effects MCF-7 and MDA-MB-231 cells. This effect appears to correlate with enhanced apoptosis in these cells. In SKBR-3 cells, mechanisms other than HSP inhibition may be utilized to a greater extent by the decoction to mediate the observed cytotoxic effects. Overall findings suggest that the decoction has the potential to be exploited further for effective treatment of breast cancer.Modulation of HSP 90 and HSP 70 expressions is a possible mechanism by which the decoction of  Flueggea leucopyrus (Willd.) also known as, Bushweed, is a medicinal plant (family: Phyllanthaceae) that grows commonly in certain regions of India, Myanmar, Pakistan and Sri Lanka . Results of the present study demonstrate that F. leucopyrus decoction can exert significant cytotoxic effects in all three different phenotypes of breast cancer cells under test. Results also demonstrate that the decoction can exert selectively greater cytotoxicity to the breast cancer cell lines than the non-cancerous breast cells MCF-10A. Thus as apparent from Fig. 50 values of 27.89, 99.43, 121.43, 570.4 μg/mL at 24 post incubation periods, for MCF-7, MDA-MB-231, SKBR-3 and MCF-10A respectively. IC50 values of the positive control paclitaxel at 24 h were 1.44, 3.47, 4.9, 2.01 μg/mL for MCF-7, MDA-MB-231, SKBR-3 and MCF-10A respectively. Higher concentrations of the decoction were used as the decoction is composed of many compounds and some of them may interact with the most cytotoxic compound of the plant. However smaller doses of paclitaxel were used, since it is a pure compound with IC50 values much lower than that of the decoction. Paclitaxel is a known chemotherapy drug used to treat different cancers including ovarian, breast and non-small cell lung cancer. It interferes with normal microtubule growth of the cell by hyper stabilising its structure [In a previous investigation Samarakoon et al.  have demtructure . The cyttructure  in Ovari50 values obtained in the SRB assay) appeared to be selectively more cytotoxic to MCF-7 cells than the other two breast cancer cell types. Overall results suggest that F. leucopyrus is selectively more cytotoxic to the Her2 negative cell lines (MCF-7 and MDA-MB-231) than to Her2 positive cell line SKBR-3. Similar results have been reported for the natural compound Curcumin [Among the three breast cancer cell phenotypes, the decoction  under investigation, but not in the SKBR-3 phenotype. This observation was apparent on evaluation of the HSP mRNA as well as HSP protein expressions in the cells by use of Real time PCR and immunofluorescence techniques respectively. Such a difference in HSP expression may be a reason for the observed differences in cytotoxicity mediated by the decoction in the MCF-7, MDA-MB-231, and SKBR-3 cells.Heat shock proteins are emerging as important molecules in cancer development and are considered to be key targets in cancer therapy. These molecular chaperones are over - expressed in many human cancers, including breast cancer , 36, 37 P <0.05) reduction in HSP 90 gene expression was observed when compared to non-treated cells (One way Anova with Dunnett’s multiple comparison test). In SKBR-3  reduction in expression of HSP 90 gene of treated MCF-7  reduction in HSP 70 gene expression was observed when compared to non-treated cells (One way Anova with Dunnett’s multiple comparison test). In SKBR-3 cells no significant effects in comparison to controls were observed in the expression of HSP 70  reduction in both MCF-7  than in MCF-7 cells although HSP70 expression was comparable in the two phenotypes. During the past decade, HSP 90 has emerged as an exciting target for cancer therapy because it has been shown to be involved in maintaining the conformation, stability, activity and cellular localization of several key oncogenic client proteins such as ERBB2, C-RAF, CDK4, AKT/PKB, steroid hormone receptors, mutant p53, HIF-1α and telomerase hTERT. It has been demonstrated to be linked to all six hall marks of cancer described by Hanahan and Weinberg [F. leucopyrus decoction than that in the poorly invasive MCF-7 cells, although effects of the decoction on HSP 70 expressions were comparable in the two phenotypes MDA-MB-231 and MCF-7.In contrast to results obtained in the cytotoxicity study, overall results of the HSP evaluation demonstrates that HSPs of the highly invasive MDA-MB-231 cells were more sensitive to the Weinberg  and moduWeinberg -40. It iApoptosis is a highly regulated process that is well characterized by distinct morphological and physiological changes in the cell. The morphological changes includes nuclear condensation, cell shrinkage and membrane blebbing, whereas the physiological changes are fragmentation of nuclear DNA due to activation of specific endonucleases cleaving nuclear DNA into 80–200 oligonucleosome fragments . These aF. leucopyrus decoction for 24 h, as indicated by alterations in cell staining (described above).Fluorescent microscopic observations of AO/EB stained Fig.  in MCF-7DNA fragmentation was observed in all three breast cancer cells exposed to the decoction for 24 h. Untreated control cells showed no evidence of DNA fragmentation while the smeared laddering pattern was observed in cells treated with 40 and 100 μg/mL of the decoction for 24 h. DNA fragmentation was done at higher doses as this event occurs at the execution phase of apoptosis . All thrP <0.05). However in MDA-MB-231 cells a statistically significant (*P <0.05) dose dependent increase in caspase activity was observed at doses <20 μg/ml  is activated in response to death stimuli such as DNA damage, chemotherapeutic agents, and UV radiation . Upon acHSP 70 or 90 binds to the Apaf-1, thereby inhibiting caspases activation and apoptotic cell death. HSP70 has also been shown to block caspase independent cell death through its association with apoptosis inducing factor (AIF) . In thisIn the present investigation it was observed that exposure of the decoction to MDA-MB-231 cells significantly increases Caspase 3/7 expression. Caspase dependent pathway otherwise known as the extrinsic pathway is another pathway that occurs in apoptosis . ExtrinsHsp90 is a homodimer, and comprises three domains. The N-terminal domain has an ATP-binding site, and natural products such as geldanamycin and radicicol binds to this domain. Co-chaperones and client proteins binds to the highly charged middle domain. ATP usually binds to the C-terminal nucleotide binding pocket but this domain has an affinity to cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol . A studyPolyalthia evecta (P. evecta) [From the overall results obtained from MCF-7 and MDA-MB-231, inhibition of HSP’s along with increase in apoptotic activity may be induced by a single bioactive compound or through a synergistic effects of different compounds present in the decoction. Anticancer effect has been reported in HepG2 cells by synergistic effects among constituents in an ethanol-water crude extract of  evecta) . However evecta) , inhibit evecta) .F. leucopyrus decoction to mediate the observed cytotoxic effects. Development of effective anti-cancer drugs has come to a halt as there is lack of phenotype specific anti-cancer agents [F. leucopyrus decoction is a promising new candidate as its cytotoxicity is significant to breast cancer cell lines and imposes minimalistic effect on normal breast cell line. Further studies are necessary in the future to identify the bioactive compounds mediating the cytotoxic and other related effects in order to determine whether a single compound or a mixture of compounds in the decoction is responsible for the observed effects.Natural anticancer compounds have been reported to mediate their actions via several mechanisms such as NFkβ inhibition, apoptosis induction, and effects on microfilament aggregation, antioxidant activity, proteasome inhibition and HSP inhibition . Howeverr agents . HSP inhr agents , 56, 57.r agents . F. leucF. leucopyrus decoction is significantly cytotoxic to all three breast cancer cell phenotypes used in this study  with greater cytotoxicity being exerted in Her2 negative phenotypes MDA-MB-231 and MCF-7 than in the Her2 positive phenotype SKBR-3. The decoction also exhibits selective cytotoxicity to the breast cancer cells in comparison with the non-cancerous breast cell line MCF-10A. These results help to rationalize the ethnopharmacological claims regarding presence of anticancer properties in F. leucopyrus. Inhibition of heat shock protein expression (HSP 70 and HSP 90) and enhanced apoptotic activity as observed in the results of this study appears to be one of the mechanisms utilized by the decoction to mediate its cytotoxic effects in the Her2 negative cell lines. In the Her2 positive SKBR-3 cells, mechanisms other than HSP inhibition appear to be utilized to a greater extent by the F. leucopyrus decoction to mediate cytotoxic effects. Overall findings of the present study suggest that the F. leucopyrus decoction has the potential to be exploited further for effective treatment of breast cancer, particularly the Her2 negative phenotypes. Confirmation of the in vitro results obtained needs to be carried out in the future by use of appropriate in vivo models.


Pinctada martensii (Pm-BMP7) was 2972 bp, including a 5'-untranslated region (UTR) of 294 bp, an open reading fragment of 1290 bp encoding a 429 amino acid polypeptide and a 3'-UTR of 1388 bp. The deduced protein sequence of Pm-BMP7 contained a signal peptide, a pro-domain and a mature peptide. The mature peptide consisted of 135 amino acids and included a transforming growth factor β family domain with six shared cysteine residues. The protein sequence of Pm-BMP7 showed 66% identity with that from Crassostrea gigas. Two unigenes encoding Pm-BMPRI (Pm-BMP receptor I) and Pm-BMPRII were obtained from the transcriptome database of P. martensii. Tissue expression analysis demonstrated Pm-BMP7 and Pm-BMPRI were highly expressed in the mantle (shell formation related-tissue), while Pm-BMPRII was highly expressed in the foot. After inhibiting Pm-BMP7 expression using RNA interference (RNAi) technology, Pm-BMP7 mRNA was significantly down-regulated (p < 0.05) in the mantle pallium (nacre formation related-tissue) and the mantle edge (prismatic layer formation related-tissue). The microstructure, observed using a scanning electron microscope, indicated a disordered growth status in the nacre and obvious holes in the prismatic layer in the dsRNA-Pm-BMP7 injected-group. These results suggest that Pm-BMP7 plays a crucial role in the nacre and prismatic layer formation process of the shell.Bone morphogenetic protein 7 (BMP7), also called osteogenetic protein-1, can induce bone formation. In this study, the obtained full-length cDNA of BMP7 from  The shell of mollusks is a stable organo-mineral product composed of calcium carbonate and organic matrix, including proteins and polysaccharides . Althoughe shell . In termet al. discovered BMPs in 1965 as active components that can induce bone formation in demineralized bone extracts [Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor β (TGF-β) superfamily. Since Urist extracts , over 20extracts . BMPs haextracts ,9, arthrextracts ,11, mollextracts ,13,14, pextracts , cnidariextracts ,17 and pextracts . StudiesPinctada martensii, also known as Pinctada fucata martensii in Japan, is the main species cultured for marine pearl production in China and Japan. It is a representative research model for biomineralized shell formation. A partial sequence of the BMP7 gene was obtained in our previous research on the transcriptome of pearl sac from P. martensii [Pm-BMP7 gene and elucidate its exact functions in shell formation.The pearl oyster artensii . The preP. martensii transcriptome database, two fragments of 210 and 1527 bp were amplified by the 5'- and 3'-RACE (rapid amplification of cDNA end) using gene-specific primers. A 2972 bp nucleotide sequence representing the complete Pm-BMP7 cDNA, was obtained by overlapping the three fragments above.Based on the 1406 bp fragment from the The complete Pm-BMP7 cDNA sequence contained a 5'-UTR of 294 bp, an open reading fragment (ORF) of 1287 bp predicted to encode a 429 amino acid polypeptide, a 3'-UTR of 1391 bp with 28 bp poly (A) tail and a typical polyadenylation signal (aataaa). This cDNA sequence has been submitted to GenBank with the Accession No. AGS32053.1. The analysis of the deduced amino acid sequence showed Pm-BMP7 contained a signal peptide (1–35 aa), a pro-domain (36–294 aa) and a mature peptide (295–429 aa). The mature peptide was produced by cleaving off the pro-domain in the putative maturation site Arg-X-X-Arg [P. martensii is homologous to the BMP7 protein of the bone morphogenetic protein family. It shared the highest identity (66%) with BMP7 from Crassostrea gigas. Meanwhile, 61% identity was shared with BMP7 from Tegillarcagranosa, 40% from Xenopus laevis, 39% from Danio rerio, and 39% from Homo sapiens.Multiple comparisons using the BLASTx program showed the deduced amino acid sequence from The homologous analysis of mature BMP7 from vertebrates and invertebrates using ClustalX software  indicated that the carboxyl terminal of Pm-BMP7, namely the TGF-β family domain, shared six cysteine residues, and the spacing between cysteine residues was well conserved among all mature BMP7 .A structure scheme of Pm-BMP7 was drawn according to homology. The six common cysteines between Pm-BMP7 and vertebrates were used to build a cystine knot by forming three intrachain disulfide bonds, whereas the additional cysteine in vertebrates could form an interchain disulfide bond .P. martensii. The unigene sequences can be found in Studies have shown that BMP7 regulates cell proliferation and differentiation by signaling through two structurally related type I (BMPRI) and type II receptors (BMPRII). Two unigenes encoding Pm-BMPRI and Pm-BMPRII were obtained in the transcriptome database of Pm-BMP7 gene on shell biomineralization in vivo, RNAi technology was applied to inhibit the expression of the Pm-BMP7 gene. The controls were RNase-free water and dsRNA-RFP injected-groups. qRT-PCR was employed to measure the mRNA levels of Pm-BMP7 in the mantle pallium, which is involved in the nacre formation, and in the mantle edge, which is involved in the prismatic layer formation 8 days after the second injection. Compared with that from the control groups, the expression of the Pm-BMP7 gene in the dsRNA-Pm-BMP7 injected group was down-regulated to approximately 22% in the mantle pallium  8 days after the second injection. The surfaces of the shells in the controls had the same normal well-defined type of microstructure. In the nacre, small hexagonal aragonite flat tablets were packed together to produce a stair-like growth pattern A,B, and C. gigas and T. granosa. Our findings raise a possibility that BMP7 in the mollusks might not form dimers or might form noncovalently linked dimers between the subunits, which could be dynamic and subject to regulation.In this report, we cloned and identified pearl oyster Pm-BMP7 and found it to be highly similar in size and sequence to BMP7 from other species. All BMPs are synthesized as large precursors, containing a signal peptide, a pro-domain and a mature peptide . This prP. martensii with the highest expression in the mantle, which is the main shell formation related-tissue. Miyashita et al. [P. martensii was predominant in the mantle by Northern blot and in situ hybridization, proposing BMP2 played a key role in the nacre formation. Iijima et al. [Lymnaea stagnalis was expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum, indicating BMP2/4 had a role in shell formation. Zoccola et al. [The tissue expression patterns showed Pm-BMP7 distributed widely in all detected tissues of a et al.  obtaineda et al.  found BMa et al.  reportedPm-BMP7 gene in shell formation, RNAi technology was used to inhibit the expression of Pm-BMP7. Compared with that from control groups, the expression of the Pm-BMP7 gene was reduced by approximately 78% in the mantle pallium and 70% in the mantle edge. Together with the SEM images of the shells, we found a disordered growth status in the aragonite tablets and obvious holes in the calcite crystals in the dsRNA-Pm-BMP7 injected group. Therefore, these results suggest that Pm-BMP7 plays a crucial role in both the nacre and prismatic layer formation process of the shell.To further elucidate the role of the 3 polymorph , the size, the shapes of the crystallites, and finally, the texture of the shell [in vivo plays a key effect on the transformation and update of mantle epidermal cells. Over expression of Pm-BMP7 and Pm-BMPRI in the mantle indicated Pm-BMP7 might be able to induce the undifferentiated cells into epidermal cells and act as an active factor to regulate shell formation. The expression of Pm-BMP7 and its receptors were considerably low in the pearl sac, which was functional in pearl formation; this phenomenon is also mentioned by Shigeharu Kinoshita [BMPs are important factors with inducing activity in the process of bone formation, and can induce mesenchymal stem cells to differentiate into osteoblasts, so as to produce new bone tissue in vertebrates . Studieshe shell . It is pinoshita . One poset al. [in vivo and in vitro have shown that the nacre [BMPs were first discovered in decalcified bone. Urist et al.  implanteet al. ,26. Intehe nacre ,28, espehe nacre ,30, can Pm-BMP7 cDNA from P. martensii, and analyzed the characteristic of its ORF and peptide sequence. Tissue expression analysis demonstrated Pm-BMP7 and Pm-BMPRI were highly expressed in the mantle, while Pm-BMPRII was highly expressed in the foot. Using RNAi technology, we have identified Pm-BMP7 participated in shell formation. To better understand how Pm-BMP7 regulates these processes, further studies on the signaling pathway are required.In conclusion, we have got the full length of Adult Pearl oysters (about 2 years of age) were obtained from Liushagang, Zhanjiang, Guangdong, China. The samples were cultured at 25–27 °C in tanks with recirculating seawater for 3–5 days before the experiment. Different tissues from the pearl oysters were acquired and immediately stored in liquid nitrogen until usage.260/OD280 with NanoDrop ND1000 spectrophotometer , while RNA integrity was determined by fractionation on a 1.0% agarose gel and staining with ethidium bromide. Total RNA (1 μg) was used as the template for the RT-reaction with M-MLV reverse transcriptase  and random primer.Total RNA was prepared using Trizol reagent  according to the manufacturer’s instructions. RNA quantity was evaluated by measuring ODBMP7 gene was obtained from the pearl sac transcriptome of P. martensii. Based on this fragment, the specific primers were designed. To increase specificity and sensitivity, nested-PCR was performed. The outer and inner primers were shown in A partial sequence of the The single-strand cDNA for all RACE reactions was prepared from the total RNA of the mantle using SMART RACE cDNA Amplification Kit  according to the manufacturer’s instructions. For the first PCR, the synthesized cDNA was used as the template. For the second PCR, the first amplicated product was used as the template. The PCR program was proceeded as follows: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 94 °C for 30 s, 68 °C for 30 s and 72 °C for 1 min, and a final elongation step of 72 °C for 10 min.Pm-BMP7 gene was analyzed using the BLAST program (http://www.ncbi.nlm.nih.gov/). The open reading fragment (ORF) was estimated using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). The amino acid sequence was characterized with DNAMAN and analyzed with expasy program (http://prosite.expasy.org/). The signal peptide was predicted by SignalP-4.0 . The mature protein molecular weight and theoretical pI were analyzed using program tool (http://web.expasy.org/cgibin/protparam/protparam). Multiple alignments were created using the ClustalX  and BioEdit softwares .The purified PCR products, containing the 5' and 3' ends, were subcloned into pMD-18T vector , transformed into DH-5α and sequenced. The full-length cDNA of The sequences of the specific primers are shown in in vivo. Sequence specific primers  for the first time, and the same doses were injected 5 days after the first injection. For the control groups, the same volume of RNase-free water and 80 μg of dsRNA-RFP in RNase-free water was separately injected each time.The PCR products were purified using EasyPure Quick Gel Extraction Kit . The dsRNA was synthesized by the T7 RNA polymerase (Thermo Scientific), and the template DNA was digested by RNase free DNase I (Thermo). The integrity and quantity of the dsRNA were verified as previously described. The dsRNA-Pm-BMP7 was diluted to 80 μg/100 µL with RNase-free water, 100 µL solutions were injected into the muscle of Pm-BMP7 gene after RNAi was measured by qRT-PCR with β-actin as the internal reference. The corresponding shells were collected, washed with Milli-Q water, air-dried, and then cut into pieces (1 cm × 1 cm). The nacre and the prismatic layer of the shells of each group were observed by an FEI Quanta 200 SEM .The total RNA was extracted from the mantle pallium and the mantle edge 8 days after the first injection and the first-strand cDNA was synthesized. The expression levels of the p-value less than 0.05 (p < 0.05) was considered as statistical significance.The data were analyzed using one-way ANOVA in SPSS 19.0 . A P. martensii. We further identified the function of Pm-BMP7 by RNAi technology. The results indicated that the obtained Pm-BMP7 in this study played a crucial role in the nacre and prismatic layer formation process of the shell. To better understand how Pm-BMP7is involved in these processes, further studies on the signaling pathways are required.In summary, we obtained the full-length cDNA of Pm-BMP7, analyzed the features of its ORF and peptide sequence, and detected the expression pattern of different tissues in


Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry. Roseobacters . Th. ThFe-Hirequency ,67,68. Ioxidases , which ilignment , we noteRd. trHb  cannot bobacters , suggestal model . The lowrin ring , a line (CβCaCb)  Table 2Fe-His inRd. trHb is 6-coordinate and low-spin with ν3 at 1496 cm-1 and ν2 at 1582 cm-1. The relatively high-frequency of the oxidation state marker ν4 at 1373 cm-1 is typical of Fe(II)CO complexes of heme proteins and is indicative of π-back-bonding from the reduced iron to CO. Even at relatively low power (less than 2 mW), a small amount of photo-dissociation of CO occurred as revealed by the detection of the ν4 (~1356 cm-1) and ν3 (1471 cm-1) lines of a 5-coordinate reduced state (FeII). These band increased in intensity at higher laser power (13 mW) and decreased at lower intensity (0.5 mW) indicating that this process was reversible (data not shown). The frequency of the ν4 line of the photo-dissociated reduced state (1356 cm-1) differed somewhat from that of the equilibrium reduced state (1362 cm-1) suggesting that only the 5-coordinate state may be present if the sixth ligand to the 6-coordinate state observed in the equilibrium sample had not formed yet.Rd. trHb (Fe-CO) was identified at 509 cm-1 (496 cm-1 with 13C18O). The frequency of the νFe-CO mode remained the same (509 cm-1) for spectra recorded at 0.5 mW (not shown) and 2 mW laser power (Rd. trHb is lower than that of wild-type Mt. trHbO (525 cm-1)  and compared the results with those of the well characterized HRP  and approximately one order of magnitude lower kcat,app (apparent kcat) and kcat,app/Km,app values compared to HRP (Rd. trHb (50 nM) was also much higher than that of bovine Hb (77.5 nM)  (Inset in Mt. trHbO (1.4 x 103 M-1s-1) and Tf. trHbO (1.9 x 103 M-1s-1). This slope is also a measure of the apparent kcat/Km when the concentration of the variable substrate is small relative to Km which reduces the Michaelis-Menten equation to V = Vmax/Km or k = kcat/Km when the initial rates are divided by the concentration of enzyme. Such an analysis gives a kcat/Km ratio of 3.0 x 104 M-1s-1 which is consistent with the kcat/Km ratio calculated from the apparent kcat and Km values  , which iubstrate ,79. To cf. trHbO ,79. ThisInset in  comparedm values . Thus, oRd. trHb, we performed steady-state kinetic measurements with H2O2/ABTS in buffers of different pHs (pH 6 to 10.5) . Data fitted with equations Ka value: 6.5 ± 0.2 , 7.0 ± 0.3  and 6.8 ± 0.2  , is observed in kcat,app, Km,app and kcat,app/Km,app. If the pKa value of ~6.8 represents the pKa of the proximal histidine of Rd. trHb, it could explain that a significant fraction of the protein displayed a proximal histidine with imidazolate character as detected from the resonance Raman spectrum collected in a buffer at pH 8.0  . The perld range . The kcigher pH . Neverth smaller . The smaKm,app) . Thus, at pH 8.0 .Rd. trHb displays clear differences of its active site compared to Mt. and Tf. trHbO, it shares with the latter a significant peroxidase activity (see above), and thus the ability to oxidize an organic substrate using hydrogen peroxide [katG gene), which has been detected in several Roseobacters [gpo gene), which is known to detoxify ROS in aerobic organisms [Rd. trHb in vivo using gene expression experiments  and the transcript levels of glbO (RD1_4240), katG (RD1_2195) and gpo (RD1_0599) were examined during the exponential growth phase. Significant up-regulation of the gpo and katG genes, along with moderate increment of glbO, were observed in the cultures exposed to H2O2, compared with cultures without H2O2, implying potential detoxification of reactive oxygen via Rd. trHb in vivo.Although peroxide . Meanwhiobacters ,82,83, mobacters ,86,87,88rganisms . Herein,eriments . R. deniM. tuberculosis was proposed to use Mt. trHbN to detoxify NO produced by macrophages [Mt. trHbO was shown to have a 1,200-fold reduced NO dioxygenase activity compared with Mt. trHbN making it unlikely that it is involved in such a reaction [Rd. trHb distinguishes this protein. However, the imidazolate character of the Fe-His stretching mode of flavohemoglobins and the single chain hemoglobin Cgb of C. jejuni, which is believed to favor their NO dioxygenase activity, is a feature shared by Rd. TrHb [Mycobacterium lepreae is involved in both H2O2 and NO scavenging [2O2 would lead to the formation of a ferryl species that would then react with NO to produce nitrite. Such a reaction bypasses the need of a reductase to reduce the heme and to allow oxygen binding as occurs in flavohemoglobins as they carry out the NO dioxygenase reaction. The relatively efficient reaction with peroxide catalyzed by Rd. trHb discussed above may be relevant to such a NO detoxification reaction.Different globins are known to possess a NO dioxygenase activity that converts NO to harmless nitrate from the reaction of the oxygenated state of these protein with NO ,89,90,91rophages , whereasreaction . It was reaction . The sinRd. TrHb . Interesavenging . For thiR. denitrificans. While aerobic denitrification is no longer recognized as an uncommon process in nature [R. denitrificans has not yet been shown to grow under strictly denitrification condition [R. denitrificans cannot grow aerobically without the supply of nitrate (data not shown), the biological and physiological roles of its aerobic denitrification activity, in addition to consuming excess reducing equivalents to minimize the production of ROS [glbO and genes involved in denitrification, biomass production and carbon assimilation are stimulated by light, whereas genes involved in carbohydrate metabolism and the TCA cycle are repressed. These observations are in agreement with the fact that Roseobacters perform photosynthesis during phototrophic growth and obtain energy from the oxidation of organic carbons via aerobic respiration during chemotrophic growth [cd1 complex) [An enzyme with a NO detoxification activity, either through a NO dioxygenase reaction or through a reaction involving a ferryl heme and NO, would be helpful during aerobic denitrification in n nature , R. deniondition  and thusn of ROS , remainsc growth . Moreovecomplex)  and acticomplex) , suggestc oxidase activity and thus could down-regulate aerobic respiration, as reported in mitochondria [R. denitrificans breaks down sugars to produce pyruvate molecules and synthesizes ATP, NADH and NADPH to preserve energy [Roseobacters have been suggested recently to be the major source of ROS in ocean [R. denitrificans produces more trHb to convert excess NO and O2 to nitrate/nitrite during phototrophic growth. A similar mechanism was also suggested recently to link the activity of THB1, which is a trHb from Chlamydomonas reinhardtii that exhibits NO dioxygenase activity, to nitrogen assimilation in this organism [NO produced by aerobic denitrification may function as a signal transducer to inhibit the cytochrome chondria . NO is achondria . The late energy . Alternae energy , which ce energy  and Rosein ocean . ROS areorganism . This hyRoseobacters. In this paper, we characterized the recombinant trHb from R. denitrificans using functional, spectral and modeling studies. The sequence alignment suggests that Rd. trHb belongs to group II trHbs and contains distal amino acids known to participate in ligand stabilization in this group of heme proteins. Resonance Raman spectra reveal a heme pocket structure consistent with the very significant peroxidase activity measured by steady-state kinetics with Rd. trHb. Together, these properties suggest a role for Rd. trHb in mechanisms of defense against reactive oxygen and/or nitrogen species or in redox biochemical reactions that remain to be explored further. Gene expression analyses also suggest that Rd. trHb contributes the detoxification of reactive oxygen and nitrogen species in vivo. Current genomic sequence data indicate that trHbs genes are widely distributed in the ecologically important marine Roseobacters. Some of the distinctive properties reported in this paper set the stage for further in-depth analyses of this interesting enzyme.Hemoglobins are known to bind various gas molecules and some of them also possess enzymatic activities. Analysis using bioinformatic tools suggest that putative globin-like proteins belonging to the truncated globin family are present in S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.